Fixed effect genetic analysis of a diallel cross in dry beans (Phaseolus vulgaris L.)

Summary A full diallel cross among four diverse homozygous strains of dry edible beans (Phaseolus vulgaris L.) was evaluated for yield, protein content, and culinary quality traits in the F₂ and F₃ generations in two locations. Interpretation of diallel effects [Method 1 Model I] using a fixed-effect genetic model made it possible to combine data from two generations into a single analysis and quantify the relative contributions of additive and dominance genetic effects to general (GCA) and specific (SCA) combining abilities. GCA was found to arise from three potential sources: additive effects, dominance interactions at homozygous loci, and average dominance interactions in hybrids involving the parent in question. SCA was found to be a function solely of dominance. Additive effects were the primary determinant of GCA and were highly significant. Specific dominance interactions were significant for seed yield, cooked bean moisture content, and texture but not for protein content. Texture was the only trait for which the additive-dominance model failed to provide an adequate fit to the data, suggesting that texture is significantly affected by epistatic interaction. One cross (Brazil-2 × Sanilac) was identified that exhibited a large heterotic effect for seed yield although the parents' additive effects were nonsignificant. Such a nicking effect was attributed to complementation between the two parents.Cooperative investigations of the Agricultural Research Service, U.S. Department of Agriculture and Michigan State University, East Lansing, MI 48824. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree at Michigan State University. Approved for publication by the Michigan Agricultural Experiment Station as Journal Article No. 11791. Research supported by USAID under a Title XII Bean/Cowpea CRSP and cooperative with Washington State University, Pullman, WA99164     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Protein inhibitors of microbial proteinases from wheat,rye and triticale

Specific protein inhibitors of microbial serine proteinases were isolated from wheat (Triticum aestivum L.), rye (Secale cereale L.) and triticale using affinity chromatography on subtilisin-Sepharose 4B. The wheat inhibitor had an isoelectric point (pI) at pH 7.2, while the rye inhibitor consisted of two forms with pI values of 6.8 and 7.1. In triticale, two components were present with pIs 7.2 and 6.8. All the inhibitors had M _r values of approx. 20 000. The isolated proteins were effective inhibitors of subtilisins Carlsberg and BPN, and of fungal proteinases (EC 3.4.21.14) from the genus Aspergillus, but they were completely inactive against trypsin (EC 3.4.21.4) chymotrypsin (EC 3.4.21.1) and pancreatic elastase (EC 3.4.21.36). The inhibitors formed complexes with subtilisin in a molar ratio of 1:1. The results of chemical modifications seem to indicate that the isolated inhibitors have methionine residues in their reactive sites.Abbreviation pI isoelectric point     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.     

Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

Cross-linking of mitochondrial matrix proteins in situ

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondrial matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase, malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the precipitates with a concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linker enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzymes are preferentially located near the membrane.     

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Effects of Preweaning Undernutrition and Continued Postweaning Protein Deficiency or Nutritional Rehabilitation on Polyphosphoinositides in Rat Brain

Metabolically inert polyphosphoinositides seem to play an important role in the structural development of neurons, glia, and myelin. The metabolically active pool of PhIpp appears to be important for the functional development of glia and myelin during the postweaning period, whereas PhIp seems to be more important for the functional development of neurons during the preweaning period. Neonatal undernutrition reduces the concentrations of structural polyphosphoinositides and metabolic PhIp while metabolic PhIpp remains unaltered. These effects can be reversed by postweaning nutritional rehabilitation. A continued postweaning protein deficiency of neonatally undernourished rats affects structural PhIpp more than PhIp. Metabolically active PhIpp is drastically reduced.